The overall goal of the research is to elucidate the mechanisms of action of enzymes that catalyze oxidations of hydrocarbons and amines. The former group includes the heme-thiolate cytochrome P450 enzymes and diiron enzymes such as methane monooxygenase and stearoyl desaturase. The latter group includes the monoamine oxidase (MAO) enzymes as well as P450 enzymes. An understanding of the mechanisms of actions of these enzymes is expected to be beneficial for therapeutic advances, for example, MAO is the target enzyme of drugs for treatment of depression and Parkinson's Disease. The method of study involves the use of "hypersensitive" radical probes that can be employed as substrates in oxidations to reveal the intermediacy of very short-lived radicals. The probe substrates are designed, and the radical reaction rate constants are determined. From product distributions in enzyme oxidations, the intermediacy of a radical is determined, and, if a radical is an intermediate, the lifetime of the radical is measured. For studies of hydrocarbon oxidations by iron-containing enzymes, some hypersensitive probes now exist, and the development of other probes is planned. Especially noteworthy is a family of probes that distinguish between radical and cationic intermediates while maintaining hypersensitive reactivity in the radical reaction. Enzyme catalyzed oxidations of calibrated probes are in progress in the PI's laboratory, using enzymes purified by other groups, and in the laboratories of other groups, using probes supplied by the PI's group. The results are expected to delineate common and dissimilar features in the mechanisms of reactions of the seemingly distinct enzymes. For studies of enzyme catalyzed amine oxidations, the work is at a more fundamental stage. Kinetic scales for reactions of a wide range of nitrogen containing radicals are being measured by a combination of direct, laser flash photolysis methods and indirect kinetic competition techniques. When the reactions of the nitrogen containing radicals are understood, rationally designed mechanistic probes will be developed and applied in oxidation reactions by P450 and MAO enzymes.